Synthetic Single Domain Antibody

ABSTRACT

The invention relates to the identification of a highly stable single domain antibody scaffold (hs2d Ab) and its use in generating synthetic single domain antibody library (hs2d Ab-L1). The invention also relates to antigen-binding proteins comprising said stable single domain antibody scaffold and their uses, in particular as therapeutics.

FIELD OF THE INVENTION

The invention relates to the identification of a highly stable syntheticsingle domain antibody scaffold and its use in generating syntheticsingle domain antibody library. The invention also relates toantigen-binding proteins comprising said stable single domain antibodyscaffold and their uses, in particular as therapeutics.

BACKGROUND OF THE INVENTION

Over the past decade antibodies imposed themselves as one of the mostpromising therapeutic approaches, in particular in the field ofoncology, as well as an important source of research or diagnosis tools.

The immunoglobulin G (IgG) is the basic structure of a typical antibody,comprising two heterodimers of heavy and light chains bond together bydisulphide bridge. Natural single chain antibodies have however beendiscovered in at least two groups of animals: Camelidae(Hamers-Casterman et al, 1993, Nature, 363, pp 446-448) and sharks(Greenberg et al, Nature. 1995, March 9; 374(6518):168-73). These singlechain antibodies constitute an additional class of IgG devoid of lightchain. The recognition part of these single chain natural antibodiesincludes only the variable domain of the heavy chain called VHH. VHHscontain four frameworks (FR) that form the scaffold of the IgG domainand three complementarity-determining regions (CDRs) that are involvedin antigen binding.

Many advantages of VHHs scaffold have been reported: without interchaindisulfide bridges, they are generally more soluble and stable in areducing environment (Wesolowski et al, 2009 Med Microbiol Immunol.August; 198(3):157-74). VHH have also been reported to have highersolubility, expression yield and thermostability due to their small size(15 kDa) (Jobling S A et al, Nat Biotechnol. 2003 January; 21 (1):77-80). Moreover, VHH frameworks show a high sequence and structuralhomology with human VH domains of family III (Muyldermans et al, 2001. JBiotechnol. June; 74 (4): 277-302) and VHHs have comparableimmunogenicity as human VH and thus constitute very interesting agentsfor therapeutic applications, some of them are currently in phase IIclinical trials (Ablynx Nanobodies®). On ten amino acids differing fromhuman, four hallmark aminoacids of VHH have been identified in theframework-2 region.

The properties of VHH scaffolds have many advantages, for use intherapy: they have a better penetration in tissues, a faster clearancein kidneys, a high specificity but also reduced immunogenicity.

Camelid antibody libraries have been described for example inUS2006/0246058 (National Research Council of Canada). The describedphage display library comprises fragments of llama antibodies, andespecially single domain fragments of variable heavy chains (VHH andVH). The libraries were made using lymphocyte genomes of non-immunizedanimals (naïve library). The resulting phage display library alsocontains contaminants of conventional VII antibody fragments.

U.S. Pat. No. 7,371,849 (Institute For Antibodies Co., Ltd) also reportsmethods of making VHH library from VHH genes of camelids. The diversityof such library was obtained by improving the conventional process ofisolating VHH variable regions from naïve repertoire.

However, these prior art do not address the issue of immunogenicity fromnon-human derived antibodies. Even if some of them are identified tobind specific target of interest, they can not be administered inpatients for use as therapeutics without the risk of activating thehuman immune system.

A method to humanize a camelid single-domain antibody is described inVincke et al, 2008, JBC Vol 284(5) pp 3273-3284.

U.S. Pat. No. 8,367,586 discloses a collection of synthetic antibodiesor their fragments. These antibodies comprise variable heavy chain andvariable light chain pairs and have, in their framework region, a partof optimal germline gene sequences. This incorporation of human sequenceallows to decrease the risk of immunogenicity for therapeutic use.

Monegal et al (2012, Dev Comp Immunol. 36(1):150-6) reports that singledomain antibodies with VH hallmarks are repeatedly identified duringbiopanning of llama naïve libraries. In fact, VH hallmarks are morefrequently identified on the binders selected from VHH naïve library,than VHH hallmarks. For example, Monegal et al have shown that 5% of VHhallmarks are round in the naïve library, while 20% of these VHhallmarks are found among the antibodies selected following biopanningagainst antigens.

Therefore, despite this knowledge, there is still a need to providesingle domain antibody libraries, with high diversity, and capable ofgenerating highly stable, humanized single domain antibody with highaffinity with a desired target.

Accordingly, one aspect of the disclosure is to provide a non-immune,recombinant single domain antibody library, of high diversity, capableof generating highly stable single domain antibody library with highaffinity against specific antigen. Another aspect is to provide alibrary enriched in single domain antibodies active in the intracellularenvironment. Yet another aspect is to provide a library enriched insingle domain antibodies with high thermostability.

SUMMARY

The purposes of the disclosure are achieved by a method of making asynthetic single domain antibody library, said method comprising thesteps of:

i) introducing a diversity of nucleic acids encoding CDR1, CDR2, andCDR3, between the respective framework coding regions of a syntheticsingle domain antibody (which may be referred to as “hs2dAb” hereafter)to generate a diversity of nucleic acids encoding synthetic singledomain antibodies with the same synthetic single domain scaffold aminoacid sequence;

wherein said synthetic single domain scaffold amino acid sequencecontains at least the following original camelid VHH amino acidresidues: F37, E44, R45, F47, and; at least the following humanizedamino acid residues: P14, S49, S74, R83, A84, and optionally furthercomprising the original camelid VHH residues Q5, Q108 and T89.

-   -   In one specific embodiment, the synthetic single domain antibody        (hs2dAb) derives from VHH of Lama species and comprises the        following humanized amino acid residues: F11, P14, S49, S74,        K75, V78, Y79, S82b, R83 and A84. In one related specific        embodiment, the synthetic single domain antibody may comprise        all the following amino acid residues: Q5, A8, F11, P14, F37,        K43, E44, R45, F47, S49, A50, S74, K75, V78, Y79, S82b, R83,        A84, T89, Q108, wherein the positions of amino acid residues are        indicated according to the Kabat nomenclature used for VH and        VHH amino acid sequence.

In one specific embodiment, the synthetic single domain antibodycomprises the following framework regions consisting of FR1 of SEQ IDNO:1, FR2 of SEQ ID NO:2, FR3 of SEQ ID NO: 3 and FR4 of SEQ ID NO:4, orfunctional variant framework regions, for example with no more than 1, 2or 3 conservative amino acid substitutions within each framework region.

In one preferred embodiment, the amino acids residues of the syntheticCDR1 and CDR2 are determined by the following rules:

-   -   at CDR1 position 1: Y, R, S, T, F, G, A, or D;    -   at CDR1 position 2: Y, S, T, F, G, T, or T;    -   at CDR1 position 3: Y, S, F, or W;    -   at CDR1 position 4: Y, R, S, T, F, G, A, W, D, E, K or N;    -   at CDR1 position 5: S, T, F, G, A, W, D, E, N, I, H, R, Q, or L;    -   at CDR1 position 6: S, T, Y, D, or E;    -   at CDR1 position 7: S, T, G, A, D, E, N, I, or V;    -   at CDR2 position 1: R, S, F, G, A, W, D, E, or Y;    -   at CDR2 position 2: S, T, F, G, A, W, D, E, N, H, R, Q, L or Y;    -   at CDR2 position 3: S, T, F, G, A, W, D, E, N, H, Q, P;    -   at CDR2 position 4: G, S, T, N, or D;    -   at CDR2 position 5: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P,        V, W, K or M;    -   at CDR2 position 6: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P,        V, W, or K;    -   at CDR2 position 7: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P,        or V;

In one related embodiment that may be combined with the precedingembodiment, said CDR3 amino acid sequence comprises between 9 and 18amino acids. In one related embodiment that may be combined with thepreceding embodiment, said CDR3 amino acid sequence comprises amino acidresidues selected among one or more of the following amino acids: S, T,F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, K, M.

The disclosure also relates to a synthetic single domain antibodylibrary obtainable by the method described above and comprising at least3·10⁹ distinct single domain antibody coding sequences.

The disclosure further concerns the use of said synthetic single domainantibody library, in a screening method, e.g. phage display, foridentifying a synthetic single domain antibody that binds to a target ofinterest, for example a human protein.

Finally, the disclosure deals with an antigen-binding protein,comprising a synthetic single domain antibody of the following formula:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein said framework regions FR1, FR2,FR3, and FR4 contains at least the following original camelid VHH aminoacid residues: F37, E44, R45, F47, and, at least the following humanizedamino acid residues: P14, S49, S74, R83, A84. Said framework regions mayfurther contain at least the following amino acid residues: Q5, Q108 andT89. In one preferred embodiment, the antigen-binding protein comprisesa synthetic single domain antibody with at least the following specificcombination of amino acid residues: Q5, A8, F11, P14, F37, K43, E44,R45, F47, S49, A50, S74, K75, V78, Y79, S82b, R83, A84, T89, Q108.

In one specific embodiment which may be combined with the precedingembodiments, the antigen-binding protein comprises a synthetic singledomain antibody having one or more of the following functionalproperties:

a) it can be expressed as soluble single domain antibody in E. coliperiplasm,

b) it can be expressed as soluble intrabodies in E. coli, yeast or othereukaryote cytosol,

c) it is stable in a reducing environment as shown in chloramphenicolacetyl transferase fusion assay,

d) it does not aggregate when expressed in mammalian cells, including asa fusion proteins (e.g. fluorescent protein fusion).

In one preferred embodiment, the framework regions of theantigen-binding protein are derived from VHH framework regions FR1, FR2,FR3, and FR4 of Lama species consisting of FR1 of SEQ ID NO:1, FR2 ofSEQ ID NO:2, FR3 of SEQ ID NO: 3 and FR4 of SEQ ID NO:4, or, theirfunctional variants, for example with no more than 0, 1, 2 or 3conservative amino acid substitutions in each of FR1, FR2, FR3 and FR4.

In another preferred embodiment, which may be combined with thepreceding embodiments, the amino acid residues of the synthetic CDR1 andCDR2 are distributed as follows:

-   -   at CDR1 position 1: Y, R, S, T, F, G, A, or D;    -   at CDR1 position 2: Y, S, T, F, G, T, or T;    -   at CDR1 position 3: Y, S, S, S, F, or W;    -   at CDR1 position 4: Y, R, S, T, F, G, A, W, D, E, K or N;    -   at CDR1 position 5: S, T, F, G, A, W, D, E, N, I, H, R, Q, or L;    -   at CDR1 position 6: S, T, Y, D, or E;    -   at CDR1 position 7: S, T, G, A, D, E, N, I, or V;    -   at CDR2 position 1: R, S, F, G, A, W, D, E, or Y;    -   at CDR2 position 2: S, T, F, G, A, W, D, E, N, H, R, Q, L or Y;    -   at CDR2 position 3: S, T, F, G, A, W, D, E, N, H, Q, P;    -   at CDR2 position 4: G, S, T, N, or D;    -   at CDR2 position 5: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P,        V, W, K or M;    -   at CDR2 position 6: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P,        V, W, or K;    -   at CDR2 position 7: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P,        or V;    -   and the CDR3 amino acid sequence comprises between 9 and 18        amino acids selected among one or more of the following amino        acids: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, K, M.

DETAILED DESCRIPTION

In the present description, positions of amino acid residues insynthetic single domain antibodies or their fragments are indicatedaccording to the Kabat numbering nomenclature as shown hereafter:

TABLE I FR or CDR VL VH FR1  1-23  1-22 CDR1 24-34   31-35B FR2 35-4936-49 CDR2 50-56 50-65 FR3 57-88 66-91 CDR3 89-97  95-102 FR4  98-107103-113

The present invention provides a method of making a synthetic singledomain antibody library, said method comprising

-   -   i. introducing a diversity of synthetic nucleic acids encoding        CDR1, CDR2, and CDR3, between the respective framework coding        regions of a synthetic single domain antibody to generate        nucleic acids encoding a diversity of synthetic single domain        antibodies with the same synthetic single domain antibody        scaffold amino acid sequence,

wherein said synthetic single domain scaffold amino acid sequencecontains at least the following original camelid VHH amino acidresidues: F37, E44, R45, F47, and; at least the following humanizedamino acid residues: P14, S49, S74, R83, A84, and optionally furthercomprising the original camelid VHH residues Q5, Q108 and T89.

The Synthetic Single Domain Antibody Scaffold of the Invention

The invention relates to the identification of unique features inframework regions of single domain antibodies, for obtaining a highlystable single domain antibody scaffold and its use in generatingsynthetic single domain antibody library, such as synthetic singledomain antibody phage display library. The resulting hs2dAb with saidunique scaffold are highly stable and have low risks of immunogenicity.

As a starting material for making the library, a nucleic acid encodingsingle domain antibody may be provided.

As used herein, the term “single domain antibody” refers to an antibodyfragment with a molecular weight of only 12-15 kDa, consisting of asingle monomeric variable antibody domain derived from a heavy chain.Said single domain antibody may derive from fragment of naturaloccurring antibodies devoid of light chains, such as so called VHHantibodies derived from camelid antibodies or so called VNAR fragmentsderived from shark species antibody. Said single domain antibody mayalso derive from human antibodies with specific following mutations:F37, E44, R45, and F47. Single domain antibody thus contains at least 4framework regions interspaced by 3 hypervariable CDR regions, resultingin the following typical antibody variable domain structure:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Said single domain does not interactwith light chain antibody variable region to form conventionalheterodimer of heavy and light chains antigen-binding VII structure.

As used herein, the term “synthetic” means that such antibody has notbeen obtained from fragments of naturally occurring antibodies butproduced from recombinant nucleic acids comprising artificial codingsequences.

In particular, the synthetic single domain antibody libraries of thedisclosure have been generated by synthesis of artificial framework andCDR coding sequences. As opposed to libraries obtained by amplificationof naïve repertoire from non-immunized llama animals, the syntheticsingle domain antibody library of the invention does not containconventional VH antibody.

Advantageously, in one preferred embodiment of the synthetic singledomain antibody library of the present invention, all single domainantibody clones contain the same framework regions, thereby providing aunique synthetic single domain antibody scaffold.

As used herein, the term “scaffold” refers to the 4 framework regions ofthe synthetic single domain antibodies of the library of the invention.Typically, all single domain antibodies of a library of the inventionhave the same scaffold amino acid sequences while their CDRs may bedifferent (the diversity of each library is only in the CDR regions).

The synthetic single domain antibody scaffold according to the presentdisclosure contains at least the following original camelid VHH aminoacid residues: F37, E44, R45, F47, and; at least the following humanizedamino acid residues: P14, S49, S74, R83, A84. Said synthetic singledomain scaffold of the present invention may optionally further comprisethe original camelid VHH residues Q5, Q108 and T89. Such unique featuresprovide highly stable synthetic single domain antibody with low risk ofimmunogenicity.

In one specific embodiment, the synthetic single domain antibodyscaffold is obtained by mutagenizing a coding sequence of VHH of Lamaspecies scaffold antibody in order to obtain at least the followinghumanized amino acid residues in the amino acid sequence: P14, S49, S74,R83, A84, and preferably, the following humanized amino acid residues:F11, P14, S49, S74, K75, V78, Y79, S82b, R83 and A84.

In one specific embodiment that may be combined with the precedingembodiment, the synthetic single domain antibody scaffold comprises

-   -   (i) a FR2 amino acid sequence that is identical to germline        Llama FR2 amino acid sequence;    -   (ii) a FR3 amino acid sequence that is identical to germline        human FR3 (VH3) amino acid sequence; and,    -   (iii) a FR4 amino acid sequence that is identical to germline        Llama FR4 amino acid sequence.

In one specific embodiment that may be combined with the precedingembodiments, the synthetic single domain antibody scaffold thuscomprises the following specific combination of amino acid residues: Q5,A8, F11, P14, F37, K43, E44, R45, F47, S49, A50, S74, K75, V78, Y79,S82b, R83, A84, T89, Q108.

In another specific embodiment, the synthetic single domain antibodyscaffold comprises the following framework regions consisting of FR1 ofSEQ ID NO:1, FR2 of SEQ ID NO:2, FR3 of SEQ ID NO: 3 and FR4 of SEQ IDNO:4, or functional variant framework regions, for example with no morethan 1, 2 or 3 conservative amino acid substitutions within eachframework region, more preferably, within only one framework region.

An example of a synthetic single domain antibody scaffold is shown inFIG. 1.

Conservative amino acid substitutions are ones in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g. lysine, arginine, histidine), acidic side chains (e.g.aspartic acid, glutamic acid), uncharged polar side chains (e.g.glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g. alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g. threonine, valine, isoleucine) and aromatic side chains(e.g. tyrosine, phenylalanine, tryptophan, histidine).

In another embodiment, the synthetic single domain antibody scaffoldcomprises functional variants of FR1, FR2, FR3 and FR4 framework regionshaving at least 90%, preferably 95% identity to SEQ ID NOs1-4respectively.

As used herein, the percent identity between two sequences is a functionof the number of identical positions shared by the sequences (i.e., %identity=# of identical positions/total # of positions×100), taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences. The comparison ofsequences and determination of percent identity between two sequencescan be accomplished using a mathematical algorithm, as described below.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Myers and W. Miller (Comput. Appl. Biosci. 4:1 1-17, 1988) which has been incorporated into the ALIGN program. Inaddition, the percent identity between two amino acid sequences can bedetermined using the Needleman and Wunsch (J. Mol. Biol. 48:443-453,1970) algorithm which has been incorporated into the GAP program in theGCG software package. Yet another program to determine percent identityis CLUSTAL (M. Larkin ef a/., Bioinformatics 23.2947-2948, 2007; firstdescribed by D. Higgins and P. Sharp, Gene 73:237-244, 1988) which isavailable as stand-alone program or via web servers (seehttp://www.clustal.org/).

Functional variants may be tested for their capacity to retain theadvantageous properties of said synthetic single domain scaffold of thepresent invention. In particular, they may be tested for their capacityto retain at least one or more of the following properties:

-   -   i. it can be expressed as soluble single domain antibody in E.        coli periplasm,    -   ii. it can be expressed as soluble intrabodies in E. coli, yeast        or other eukaryote cytosol,    -   iii. it is stable in reducing environment in chloramphenicol        acetyl transferase fusion assay,    -   iv. it does not aggregate when expressed in mammalian cells,        including as a fusion proteins (e.g. fluorescent protein        fusion).

Assays for testing the above properties are described in the Examples.

For example, a reference synthetic single domain antibody codingsequence is constructed by grafting reference CDRs coding sequences(such as the CDRs of clone D10 of SEQ ID NO:9) into a variant scaffoldcoding sequence to be tested (with homologous sequences to SEQ ID NOs1-4). This reference synthetic single domain antibody coding sequenceallows to produce a reference synthetic single domain antibody which canbe assayed for the above properties.

Introduction of CDR Diversity in the Selected Single Domain AntibodyScaffold

Methods for generating CDRs diversity for antibody libraries, inparticular by random or directed synthesis of CDR coding sequences andcloning into corresponding framework sequences have been widelydescribed in the art.

The synthetic single domain antibody libraries of the present disclosureare generated similarly by introducing CDR high diversity into theunique selected scaffold sequence, for example, as described in Lindner,T., H. Kolmar, U. Haberkorn, and W. Mier. 2011. Molecules. 16:1625-1641.

In one preferred embodiment of the present disclosure, the position ofeach amino acid sequence of synthetic CDR1 and CDR2 is rationallydesigned to mimic natural diversity of CDRs in human repertoire.

Cysteines are voluntarily avoided because of their thiol groups whichmay interfere with intracellular expression and functionality. Besides,arginine and hydrophobic residues may also be avoided because of thehigh risk aggregation of the resulting antibody. A low proline rate isalso preferred because it provides more flexibility in the CDRs.Preferably, serine, threonine and tyrosine are the most frequentresidues in all three CDRs, as being involved in bonds with the epitope.Aspartate and glutamate may also be enriched at some positions in orderto increase solubility. For CDR3 sequences, the lengths may influencethe binding potential to different epitope shape, in particular cavity.Therefore, different lengths of CDR3 sequences may be introduced intothe libraries.

In one specific embodiment, the skilled person may select the amino acidresidues of the synthetic CDR1 and CDR2 according to the followingrules:

-   -   at CDR1 position 1: Y, R, S, T, F, G, A, or D;    -   at CDR1 position 2: Y, S, T, F, G, T, or T;    -   at CDR1 position 3: Y, S, F, or W;    -   at CDR1 position 4: Y, R, S, T, F, G, A, W, D, E, K or N;    -   at CDR1 position 5: S, T, F, G, A, W, D, E, N, I, H, R, Q, or L;    -   at CDR1 position 6: S, T, Y, D, or E;    -   at CDR1 position 7: S, T, G, A, D, E, N, I, or V;    -   at CDR2 position 1: R, S, F, G, A, W, D, E, or Y;    -   at CDR2 position 2: S, T, F, G, A, W, D, E, N, H, R, Q, L or Y;    -   at CDR2 position 3: S, T, F, G, A, W, D, E, N, H, Q, P;    -   at CDR2 position 4: G, S, T, N, or D;    -   at CDR2 position 5: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P,        V, W, K or M;    -   at CDR2 position 6: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P,        V, W, or K;    -   at CDR2 position 7: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P,        or V;

Furthermore, in another specific embodiment, CDR3 amino acid sequencecomprises between 9 and 18 amino acids selected among one or more of thefollowing amino acids: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V,W, K, M.

The above rules of occurrence are used as a guidance for generatingpreferred libraries of the invention, however, other libraries withdifferent occurrence rules are also part of the invention, as long asthey contain the advantageous synthetic single domain antibody scaffoldof the present invention.

In specific embodiments, only a significant proportion of the clones ofthe library may follow strictly the above rules of occurrence. Forexample, statistically, at least 50%, 60%, 70%, 80% or at least 90% ofthe clones of the library follow the above rules of occurrence of aminoacid residues in CDR1, CDR2 and CDR3 positions.

In order to respect these occurrences of amino acid positions, and toavoid the occurrence of in frame stop, or cysteine or reduce frameshift,advanced gene synthesis approaches are preferably used. These methodsencompass, but are not limited to, double strand DNA triple blocks asdescribed in Van den Brulle et al., 2008. Biotechniques 45(3): 340-3,tri-nucleotide synthesis, or other codon-controlled and more generallyposition-controlled degenerate synthesis approaches.

In specific embodiments, codon bias may further be optimized for examplefor host cell species, for example, mammalian host cells expression,using well known methods.

In one specific embodiment, the coding sequence is designed so that itdoes not contain undesired restriction sites, for example, restrictionsites that are used for cloning the coding sequence into the appropriatecloning or expression vector.

The resulting diverse coding sequences are introduced into a suitableexpression or cloning vectors for antibody libraries. In a specificembodiment, the expression vector is a plasmid. In another preferredembodiment, the expression vector is suitable for generating phagedisplay libraries. Two different types of vectors may be used forgenerating phage display libraries: phagemid vectors and phage vectors.

Phagemids are derived from filamentous phage (Ff-phage-derived) vectors,containing the replication origin of a plasmid. The basic components ofa phagemid mainly include the replication origin of a plasmid, theselective marker, the intergenic region (IG region, usually contains thepacking sequence and replication origin of minus and plus strands), agene of a phage coat protein, restriction enzyme recognition sites, apromoter and a DNA segment encoding a signal peptide. Additionally, amolecular tag can be included to facilitate screening of phagemid-basedlibrary. Phagemids can be convened to filamentous phage particles withthe same morphology as Ff phage by co-infection with the helper phages,such as R408, M13KO7 and VCSM13 (Stratagene). One example of phagevector is fd-tet (Zacher et al, gene, 1980, 9, 127-140) which consistsof fd-phage genome and a segment of Tn10 inserted near the phage genomeorigin of replication. Examples of promoters for use in phagemid vectorsinclude, without limitation, PlacZ or PT7, examples of signal peptideincludes without limitation pelB leader, gill, CAT leader, SRP or OmpAsignal peptide.

Other phage-display methods use lytic phages like T4 or T7. Vectorsother than phages may also be used to generate display libraries,including vectors for bacterial cell display (Daugherty et al., 1999Protein Eng. July; 12(7):613-21, Georgiou et al., 1997 Nat Biotechnol.1997 January; 15(1):29-34), yeast cell display (Boder and Wittrup, NatBiotechnol. 1997 June; 15(6):553-7) or ribosome display (Zahnd C.Amstutz P. Plückthun A. Nat Methods. 2007 March; 4(3):269-79). DNAdisplay (Eldridge et al., Protein Engineering. Design & Selection vol.22 no. 11 pp. 691-698, 2009) and surface display on mammalian cells(Rode H J, et al. Biotechniques. 1996 October; 21(4):650, 652-3, 655-6,658) have also been reported. Non display methods like yeast two-hybridmay also be used to select relevant binders from the library (Visintinet al., 1999 Proc Natl Acad Sci USA 96, 11723-11728.).

In one preferred embodiment, in order to avoid generating empty vectors,positive selection of recombinant coding sequence in the cloning vectorsbearing a suicide gene is applied (see for example Philippe Bernard,1996, BioTechniques, Vol 21, No 2 “Positive Selection of Recombinant DNAby CcdB”).

Preferably, the theoretical diversity as calculated by all possiblecombination of CDR amino acid residues as designed for generating theantibody library is at least 10¹¹ or at least 10¹¹ unique sequences.

Synthetic Single Domain Antibody Library of the Invention and their Use

Consequently, according to another aspect, the disclosure relates to asynthetic single domain antibody library obtainable or obtained by theprevious method.

As used herein, the term “synthetic single domain antibody library” thusencompasses nucleic acid libraries comprising said synthetic singledomain antibody coding sequences with high diversity, optionallyincluded in a cloning vector or expression vector. The term “syntheticsingle domain antibody library” further includes any transformed hostcells or organisms, with said nucleic acid libraries, and morespecifically, bacterial, yeast or filamentous fungi, or mammalian cellstransformed with said nucleic acid libraries, or bacteriophages orviruses containing said nucleic acid libraries. The term “syntheticsingle domain antibody library” further includes the correspondingmixture of diverse antibodies encoded by said nucleic acid library. Asused herein, the term “clone” will refer to each unique individual ofthe antibody library, whether, nucleic acids, host cells, or singledomain antibodies.

In one specific embodiment of the disclosure, the synthetic singledomain antibody library of the present disclosure comprises at least3·10⁹ diverse clones.

This library may be used in a screening method, for identifying asynthetic single domain antibody that binds specifically to a target ofinterest. Any known screening methods for identifying binders withspecific affinity to a target of interest may be used with the syntheticsingle domain antibody libraries of the invention. Such methods includewithout limitation phage display technologies, bacterial cell display,yeast cell display, mammalian cell display or ribosome display.

Preferably, the screening method is the phage display.

Preferably, the target of interest is a therapeutic target, and thesynthetic single domain antibody library is used to identify syntheticsingle domain antibody with specific binding to said therapeutic target.In specific embodiments, the target of interest comprises at least anantigenic determinant. In specific embodiments, the target is asaccharide or polysaccharide, a protein or glycoprotein, a lipid. In onespecific embodiment, said target of interest is of plant, yeast, fungus,insect, mammalian or other eukaryote cell origins. In another specificembodiment, said target of interest is of bacterial, protozoan or viralorigin.

In one specific embodiment. “a single domain antibody that bindsspecifically to a target of interest” is intended to refer to singledomain antibody that binds to the target of interest with a K_(D) of 1mM or less. 100 μM or less. 101 μM or less. This does not exclude thatsaid single domain antibody also binds to other antigens.

The term “K_(D)”, as used herein, is intended to refer to thedissociation constant, which is obtained from the ratio of K_(d) toK_(a) (i.e. K_(d)/K_(a)) and is expressed as a molar concentration⁻¹(M⁻¹). K_(D) values for antibodies can be determined using methods wellestablished in the art. A method for determining the K_(D) of anantibody is by using surface plasmon resonance, or using a biosensorsystem such as a Biacore® system or Proteon®.

Antigen-Binding Protein of the Invention

Considering the high diversity of the synthetic single domain antibodylibraries of the invention, the skilled person can obtain syntheticsingle domain antibody with high affinity and high specificity to atarget of interest, by conventional screening methods, such a phagedisplay.

The resulting synthetic single domain antibody can then be furthermodified for generating appropriate antigen-binding protein. Inparticular, the CDR residues may be modified for example to increase theantibody affinity to the target of interest, improve its folding or itsproduction, using technologies known in the art (mutagenesis, affinitymaturation).

Accordingly, another aspect of the invention further relates to anantigen-binding protein, comprising a synthetic single domain antibodyof the following formula: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein saidframework regions FR1, FR2, FR3, and FR4 contains at least the followingoriginal camelids VHH amino acid residues: F37, E44, R45, F47, and; atleast the following humanized amino acid residues: P14, S49, S74, R83,A84.

In one embodiment, the framework regions further contain at least thefollowing amino acid residues: Q5, Q108 and T89. In another embodiment,the synthetic single domain antibody comprises the specific combinationof at least the following amino acid residues: Q5, A8, F11, P14, F37,K43, E44, R45, F47, S49, A50, S74, K75, V78, Y79, S82b, R83, A84, T89,Q108.

In one preferred embodiment, the framework regions are derived from VHHframework regions FR1, FR2, FR3, and FR4 of Lama species. In anotherspecific embodiment, the single domain antibody scaffold comprises

-   -   (i) a FR2 amino acid sequence that is identical to germline        Llama FR2 amino acid sequence;    -   (ii) a FR3 amino acid sequence that is identical to germline FR3        (V113) amino acid sequence; and,    -   (iii) a FR4 amino acid sequence that is identical to germline        Llama FR4 amino acid sequence.

In one preferred embodiment, the synthetic single domain antibodycomprises either of the following features:

-   -   (i) framework regions FR1 of SEQ ID NO:1, FR2 of SEQ ID NO:2,        FR3 of SEQ ID NO:3, and FR4 of SEQ ID NO:4,    -   (ii) functional variant framework regions having no more 1, 2 or        3 amino acid conservative substitutions and retaining        advantageous synthetic single domain properties,    -   (iii) functional variant framework regions FR1, FR2, FR3 and FR4        having at least 60, 70, 80, 90, 95, 96, 97, 98, or 99 percent        sequence identity to SEQ ID NOs:1-4 respectively and retaining        advantageous synthetic single domain properties.

Typically, one or more amino acid residues within the framework regionscan be replaced with other amino acid residues from the same side chainfamily, and the new polypeptide variant can be tested for retainedadvantageous properties using the functional assays described herein.

Such advantageous properties are one or more of the followingproperties:

-   -   i. it can be expressed as soluble single domain antibody in E.        coli periplasm

Typically, a yield exceeding 5 mg/L with a pelB leader peptide may bepreferably obtained in E. coli strains.

-   -   ii. it can be expressed as soluble intrabodies in E. coli        cytosol

For example, antibodies may be expressed in E. coli strains BL21(DE3) ata yield exceeding 50 mg/liter with a T7 promoter.

-   -   iii. it is stable in a reducing environment as shown in        chloramphenicol acetyl transferase fusion assay

Functional assays for the above properties are described in theExamples. The bacterial cells expressing said antigen-binding proteincontaining the synthetic single domain antibody of the invention infusion with chloramphenicol acetyl transferase as a C-terminal tagshould be resistant to chloramphenicol, in particular, at aconcentration of chloramphenicol higher than 300 μg/ml in culturemedium.

-   -   iv. it does not aggregate when expressed in mammalian cell lines        as fluorescent protein fusions.

Preferably, no aggregation should be detected when the antigen-bindingprotein containing the synthetic single domain antibody is expressed asfluorescent protein fusion.

Preferably, the amino acid residues of the synthetic CDR1 and CDR2 maybe:

-   -   at CDR1 position 1: Y, R, S, T, F, G, A, or D;    -   at CDR1 position 2: Y, S, T, F, G, T, or T;    -   at CDR1 position 3: Y, S, S, S, F, or W;    -   at CDR1 position 4: Y, R, S, T, F, G, A, W, D, E, K or N;    -   at CDR1 position 5: S, T, F, G, A, W, D, E, N, I, H, R, Q, or L;    -   at CDR1 position 6: S, T, Y, D, or E;    -   at CDR1 position 7: S, T, G, A, D, E, N, I, or V;    -   at CDR2 position 1: R, S, F, G, A, W, D, E, or Y;    -   at CDR2 position 2: S, T, F, G, A, W, D, E, N, H, R, Q, L or Y;    -   at CDR2 position 3: S, T, F, G, A, W, D, E, N, H, Q, P;    -   at CDR2 position 4: G, S, T, N, or D;    -   at CDR2 position 5: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P,        V, W, K or M;    -   at CDR2 position 6: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P,        V, W, or K;    -   at CDR2 position 7: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P,        or V;

and CDR3 amino acid sequence comprises between 9 and 18 amino acidsselected among one or more of the following amino acids: S, T, F, G, A,Y, D, E, N, I, H, R, Q, L, P, V, W, K, M.

Accordingly, in one preferred embodiment, the antigen-binding protein ofthe invention, essentially consists of a synthetic single domainantibody of the general formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In suchembodiment, more preferably, FR1 is SEQ ID NO:1, or a functional variantof SEQ ID NO:1 with 1, 2 or 3 amino acid substitutions. FR2 is SEQ IDNO:2, or a functional variant of SEQ ID NO:2 with 1, 2 or 3 amino acidsubstitutions; FR3 is SEQ ID NO:3, or a functional variant of SEQ IDNO:3 with 1, 2 or 3 amino acid substitutions; FR4 is SEQ ID NO:4, or afunctional variant of SEQ ID NO:4 with 1, 2 or 3 amino acidsubstitutions; CDR1, CDR2 amino acid sequences have amino acid residuesas follows:

-   -   at CDR1 position 1: Y, R, S, T, F, G, A, or D;    -   at CDR1 position 2: Y, S, T, F, G, T, or T;    -   at CDR1 position 3: Y, S, S, S, F, or W;    -   at CDR1 position 4: Y, R, S, T, F, G, A, W, D, E, K or N;    -   at CDR1 position 5: S, T, F, G, A, W, D, E, N, I, H, R, Q, or L;    -   at CDR1 position 6: S, T, Y, D, or E;    -   at CDR1 position 7: S, T, G, A, D, E, N, I, or V;    -   at CDR2 position 1: R, S, F, G, A, W, D, E, or Y;    -   at CDR2 position 2: S, T, F, G, A, W, D, E, N, H, R, Q, L or Y;    -   at CDR2 position 3: S, T, F, G, A, W, D, E, N, H, Q, P;    -   at CDR2 position 4: G, S, T, N, or D;    -   at CDR2 position 5: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P,        V, W, K or M;    -   at CDR2 position 6: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P,        V, W, or K;    -   at CDR2 position 7: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P,        or V;    -   and CDR3 amino acid sequence comprises between 9 and 18 amino        acids selected among one or more of the following amino acids:        S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, K, M.

Another aspect of the disclosure pertains to nucleic acid molecules thatencode the antigen-binding proteins of the disclosure. The disclosurethus provides an isolated nucleic acid encoding at least said syntheticsingle domain antibody portion of the antigen-binding protein.

The nucleic acids may be present in whole cells, in a cell lysate, ormay be nucleic acids in a partially purified or substantially pure form.A nucleic acid is “isolated” or “rendered substantially pure” whenpurified away from other cellular components or other contaminants, e.g.other cellular nucleic acids or proteins, by standard techniques,including alkaline/SDS treatment, CsCI banding, column chromatography,agarose gel electrophoresis and others well known in the art. See, F.Ausubel, et al., ed. 1987 Current Protocols in Molecular Biology, GreenePublishing and Wiley Interscience, New York. A nucleic acid of theinvention can be, for example, DNA or RNA and may or may not containintronic sequences.

In an embodiment, the nucleic acid is a DNA molecule. The nucleic acidmay be present in a vector such as a phage display vector, or in arecombinant plasmid vector. In one specific embodiment, the inventionthus provides an isolated nucleic acid or a cloning or expression vectorcomprising at least one or more of the following nucleic acid sequences:SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, encodingrespectively framework regions FR1, FR2, FR3 and FR4 of SEQ ID NOs 1-4,or variant corresponding sequences with at least 90% identity to saidSEQ ID NOs 5-8, encoding functional variants of FR1, FR2, FR3, and FR4of SEQ ID NOs 1-4.

DNA fragments encoding the antigen-binding proteins, as described aboveand in the Examples, can be further manipulated by standard recombinantDNA techniques, for example to include any signal sequence forappropriate secretion in expression system, any purification tag andcleavable tag for further purification steps. In these manipulations, aDNA fragment is operatively linked to another DNA molecule, or to afragment encoding another protein, such as a purification/secretion tagor a flexible linker. The term “operatively linked”, as used in thiscontext, is intended to mean that the two DNA fragments are joined in afunctional manner, for example, such that the amino acid sequencesencoded by the two DNA fragments remain in-frame, or such that theprotein is expressed under control of a desired promoter.

The antigen-binding proteins of the disclosure can be produced in a hostcell transfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art.For expressing and producing recombinant antigen-binding proteins of theinvention in host cell transfectoma, the skilled person canadvantageously use its own general knowledge related to the expressionand recombinant production of antibody molecules or single domainantibody molecules.

The disclosure thus provides a recombinant host cell suitable for theproduction of said antigen-binding proteins of the invention, comprisingthe nucleic acids, and optionally, secretion signals. In a preferredaspect the host cell of the invention is a mammalian cell line. Theinvention further provides a process for the production of anantigen-binding protein, as described previously, comprising culturingthe host cell under appropriate conditions for the production of theantigen-binding protein, and isolating said protein.

Mammalian host cells for secreting the antigen-binding proteins of thedisclosure, include CHO, such as dhfr-CHO cells, (described by Urlauband Chasin, 1980, Proc. Natl. Acad. Sci. USA 77:4216-4220) used with aDHFR selectable marker, e.g. as described in R. J. Kaufman and P. A.Sharp, 1982 Mol. Biol. 159:601-621, NSO myeloma cells, or the pFuseexpression system from Invivogen, as described in Moutel, S., El Majou,A., Vielemeyer, O., Nizak, C., Benaroch, P., Dubel, S., and Perez, F.(2009). A multi-Fc-species system for recombinant antibody production.BMC Biotechnol 9, 14, COS cells and SP2 cells or human cell lines(including PER-C6 cell lines, Crucell or HEK293 cells, Yves Durocher etal., 2002, Nucleic acids research vol. 30, No 2 p9). When said nucleicacids encoding antigen-binding proteins of the invention are introducedinto mammalian host cells, the antigen-binding proteins are produced byculturing the host cells for a period of time sufficient to allow forexpression of the recombinant polypeptides in the host cells orsecretion of the recombinant polypeptides into the culture medium inwhich the host cells are grown and proper refolding to produce saidantigen-binding proteins.

The antigen-binding protein can then be recovered from the culturemedium using standard protein purification methods.

In one specific embodiment, the present disclosure provides multivalentantigen-binding proteins of the invention, for example in the form of acomplex, comprising at least two identical or different synthetic singledomain antibody amino acid sequences of the invention. In oneembodiment, the multivalent protein comprises at least two, three orfour synthetic single domain antibody amino acid sequences. Thesynthetic single domain amino acid sequences can be linked together viaprotein fusion or covalent or non-covalent linkages.

In another aspect, the present disclosure provides a composition. e.g. apharmaceutical composition, containing one or a combination of theantigen-binding proteins of the present invention, formulated togetherwith one or more pharmaceutically acceptable vehicles or carriers.

Pharmaceutical formulations of the disclosure may be prepared forstorage by mixing the proteins having the desired degree of purity withoptional physiologically acceptable carriers, excipients or stabilizers(Remington: The Science and Practice of Pharmacy 20th edition (2000)),in the form of aqueous solutions, lyophilized or other driedformulations.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas, aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage.

In the following, the disclosure will be illustrated by means of thefollowing examples and figures.

FIGURES LEGENDS

FIG. 1A-C. (A). Chloramphenicol acetyl transferase carboxy terminalfusion is a folding reporter allowing the selection of soluble aminoterminal VHH. Scheme of the construct expressed from pAO-VHH-CATHavector (B) Relative colony growth of selected VHH on chloramphenicolselection medium (Cam). (C) Amino acid sequences alignment of the lamascaffold IsdAb with the humanized synthetic scaffold of the libraryhs2dAb. Positions (IMGT numbering) were highlighted according to aminoacids conserved from the D10 intrabody scaffold for intrinsic solubilityproperties of VHH (black) and humanized residues conserved with humanconventional VH3 (Grey).

FIG. 2A-D (A) Phages presenting both scaffold were produced in E. coliand supernatant were detected in Western Blot with an anti-pIII antibody(Biolabs). Two bands are visible, one for pill and one for the fusionwith single domains. (B) Production of both scaffolds in E. coli or inCHO cells analysed by Dot Blot. Serial dilutions of supernatant wererevealed with an anti-HisTag antibody (Sigma). (C) Immunofluorescence ofHeLa cells with recombinant Ab from both scaffolds labelling the nuclearrim structure characteristic of the nuclear lamina. (D) HeLa cells weretransiently transfected with the GFP-fused anti-Lamin antibodiesexpression plasmids and live cells were imaged after 24 h. This showedthat both synthetic antibody scaffold anable the recognition of theintracellular lamin target.

FIG. 3A-C (A) s2dAb D5 stained microtubules by immunofluorescence inHeLa cells. Cells were fixed and stained with a VHH revealed by ananti-HisTag (Sigma) and an anti-MouseCy3 secondary antibody (Jackson).D5 detect also tubulin in Western blot experiment with HeLa cellextracts revealed with HRP secondary antibody (Jackson). (B) Phage ELISAof clone D10 anti-Her2 on Her2 fused with a rabbit Fc versus binding onrabbit Fc at equimolar concentration. FACS analysis of HD10 anti-Her2 onSKBR3 Her2 positive cells versus MCF10A Her2 negative cells. (C) H12 isa conformational antibody binding only to the GTP bound, activated stateof RhoA GTPase. A CBD tagged H12 pull down from HeLa cell extract loadedwith either 100 μM GTP gamma S (GTP) or with 1 mM GDP as inputs. Westernblot experiment reveals RhoA at similar level in 5% of both input butonly on the GTP loaded extract in the CBD-H12 pull down. D5 anti tubulinis a negative control and the conventional GST-RBD (Rho binding domainof Rhotekin) is shown as a positive control of active Rho pull down.

FIG. 4A-C Intracellular expression of hs2dAb. (A) HeLa cells werecotransfected with GFP tagged Rab6 and mCherry-tagged hs2dAb anti-GFPplasmids. The hs2dAb-mCherry anti-GFP interacted with its target in vivoand the mCherry signal colocalized perfectly with the GFP-Rab6 one. (B))HeLa cells were cotransfected with Myr-palm-mCherry and a hs2dAb-GFPanti-mCherry plasmids. The hs2dAb-GFP anti-mCherry interacted with itstarget in vivo and colocalized perfectly with the mCherry localized atthe plasma membrane. (C) Hela cells (p53+/+) and U2OS (p53−/−) cellswere transfected either with a hs2dAb-mCherry anti-p53 antibody or witha hs2dAb-mCherry anti-Lamin. While the anti-Lamin intrabody labelledboth cells types, the anti-p53 intrabody labelled only the nuclei ofp53-positive cells.

FIG. 5 Sensorgrams for the binding of hs2dAb anti-GFP, anti-p53 andanti-Her2 on immobilized antigens. Different concentrations of hs2dAbwere loaded at 25° C. and fitted with a 1:1 langmuir interaction model.

EXAMPLES

Functional Assays

Soluble Expression in E. coli Periplasnm

Single domain antibody fragments were subcloned in a pHEN6 derivatedbacterial periplasm expression vector and expressed downstream of thepelB secretion sequence. Freshly transform colonies were grown inTerrific Broth medium supplemented with 1% glucose and 30 μg/mlkanamycin antibiotic until A600=0.6-0.8 was reached. The expression ofantibody fragment tagged with 6 His was then induced with 500 μMisopropyl β-D-thiogalactopyranoside for 16 h at 28° C. then span down.After centrifugation, the cell pellets were incubated inTris-EDTA-Sucrose osmotic shock buffer and centrifuged again. The celllysates were cleared and loaded onto an IMAC resin affinity column forpoly Histidine tag. The eluted fraction was dialyzed, and the purity ofthe protein was analyzed by SDS-PAGE.

Soluble Expression of Intrabodies in E. coli Cytosol

Single domain antibody fragments were subcloned in a bacterialexpression vector under the control of a T7 promoter. The plasmidconstructs were transformed into E. coli BL21(DE3) cells. Singlecolonies were grown in LB medium supplemented with 1% glucose and 30μg/ml kanamycin antibiotic until A600=0.6-0.8 was reached. Antibodyfragment expression was then induced with 500 μM isopropylβ-D-thiogalactopyranoside for 6 h to 8 h at 28° C. then span down. Aftercentrifugation, the cell pellets were lysed and centrifuged again. Thecell lysates were cleared and loaded onto an IMAC resin affinity columnfor poly Histidine tag. The eluted fraction was dialyzed, and the purityof the protein was analyzed by SDS-PAGE.

Chloramphenicol Acetyl Transferase Fusion Assay

Single domain antibody fragments were subcloned in the pAOCAT bacterialperiplasm expression vector. Chloramphenicol resistance assay wasperformed using BL21(DE3) cells transformed with the pAOCAT-VHH fusionconstructs. Bacteria were used for inoculating 500 μL of LB containingkanamycin (35 μg/mL) and glucose (0.2%), and were grown at 37° C. untilOD600 was 0.8. The cytoplasmic expression of the VHH-CAT-fusion proteinswas induced for 2 hours by the addition of 0.2 mM IPTG. At the end ofthe induction period, bacteria aliquots of 4 μL were plated on LB-agarplates containing IPTG (0.1 mM) and increasing chloramphenicolconcentrations ranging from 0 to 500 μg/ml. Bacteria were incubated at30° C. for 20 hours before quantification of the colony formation. Theresistance level was evaluated according to the colony growth rate atthe different chloramphenicol concentrations. Several VHH that weregiving colonies up to 500 μg/ml were compared to previouslycharacterized intrabodies raised against GFP (nb GFP4) or Lamin (Lam) aswell as to a thermostable VHH Re3 and a non intrabody C8. Liquid cultureinduced as above during 2 hours were diluted by serial dilution and 10μl were spotted on agar plates containing 250 μg/ml chloramphenicol(Cam) and incubated at 30° C. for 20 hours. Colony were quantified foreach dilution and normalized to the higher amount always found with theD10 clone.

Aggregation Assays in Mammalian Cell Expression System

Functional Expression as Intracellular Antibodies in Eukaryote Cells

Single domain antibody fragments were subcloned into a mammalianexpression vector in order to express it as a fusion with a fluorescentprotein and under the control of a CMV promoter. Mammalian cell lineswere transfected and fluorescence in the cells was observed 24 h or 48 hafter transfection. In comparison to non fused GFP or mCherry, thefluorescence distribution of VHH fused to one of these fluorescentproteins was homogenously spread in transfected cells, showing noobvious aggregates after 48 h of constitutive expression.

Functional Secretion as Fc Fusion

The plasmids are based on the pFUSE-Fc2(IL2ss)™ series from Invivogen(San Diego, USA) that contains the interleukin-2 (IL2) signal sequenceand allows the secretion of Fc-Fusion proteins by mammalian cells.Because the hs2dAbs were fused to the hinge domain of IgGs, the Fcdomains form di-sulfide bridges and hs2dAb-Fc are expressed as dimmers.They are selectable using Zeocin™ (Zeo) both in prokaryotic andeukaryotic cells. These plasmids were modified by site directedmutagenesis and adaptor insertion (Mould S, et al. BMC Biotechnol. 2009Feb. 26; 9:14. doi: 10.1186/1472-6750-9-14) to allow the easy one stepcassette cloning of recombinant antibodies extracted from a largecollection of common recombinant antibody selection and expressionplasmids (e.g pHEN, pSEX, pHAL, pCANTAB, pHOG, pOPE, pSTE). Fourplasmids were constructed enabling fusion of s2dAb at their C terminuswith either human IgG2 (and IgG1) (h), mouse IgG2a (m) or the rabbit IgG(r) Fc domain (Fc regions comprise the CH2 and CH3 domains of the IgGheavy chain and the hinge region).

Four days after transient transfection of CHO or HEK cells with theseexpression plasmids, secreted antibodies could be available usinganti-IgG antibodies directed against the respective Fc species. Thisallows a large diversity of multiplexing.

Functional Expression in Yeast Two Hybrid System

Antigen coding sequence was cloned in yeast two hybrid bait plasmid lexA(Vojtek and Hollenberg (1995). Methods Enzymol. 255:331-42).

or gal4 (Fromont-Racine, M., Rain, J. C., and Legrain, P. (1997). Nat.Genet. 16: 277-282). DNA binding domain SDAB population to be tested wastransferred in yeast two hybrid prey plasmid pGADGH (Bartel, P. L., etal (1993) in Cellular interactions in development: A practical approach.ed. Hartley. D. A. (Oxford University Press, Oxford) pp. 153-179.) byPCR and Gap repair (Orr-Weaver. T. L. and Szostak, J. W. (1983). Proc.Natl. Acad. Sci. USA 80, 4417-4421): DNA prep of pHEN2-3myc plasmid pool(from 1 single clone to 3.109) was prepared. The miniprep DNA wasamplify by PCR with oligonucleotide 5p 8328CCCACCAAACCCAAAAAAAGAGATCCTAGAACTAGCTATGGCCGGACGGGCCATGGCGGAAGTGCAGCTGCAGGCTTC (SEQ ID NO:11) and oligonucleotide 3p 8329ACCGGGCCTCTAGACACTAGCTACTCGAGGGGCCCCAGTGGCCCTATCTATGCGGCCGCGCTACTCACAGTTAC (SEQ ID NO:12) using pFu polymerase (NEB) using 10ng of DNA as matrix. The number of tube is depending of the need in DNAquantity and is related to the number of transformant needed. Typicallyto obtain 1 million yeast transformants, we carried out 8 PCR of 50 μl.

PCR program 45 secondes 94° C. {close oversize brace} ×25 cycles 45secondes 94° C. 45 secondes 57° C.  3 minutes 72° C. 10 minutes 72° C. ∞ 4° C.

PCRs are checked on an agarose gel and concentrated 20 times usingammonium acetate precipitation and resuspended in water.

8 μg of plasmid prey pGADGH digested by NcoI and XhoI and 2 μl ofconcentrated PCR are transformed in yeast by classical LiAc/PEGtransformation. The clones are spread to selective media dropt out minusTryptophane, Leucine and Histidine. The baits specific clones,identified by this way, are intrabodies from the library that arefunctional in the yeast cells.

Generation of Synthetic Single Domain Antibody Library andCharacterization of Binders Obtained from Said Library

We selected a family of highly functional scaffolds, optimized forintracellular expression and high thermostability. This selection wasdone using fusion proteins between an antibiotic resistance gene and acollection of VHH.

Only bacteria expressing a functional VHH fusion (non aggregating, nondegraded) could grow. Expression yield, solubility as GFP fusion inmammalian cells cytoplasm have been further assessed [and compared toselected chromobodies] to select a set of suitable antibodies. Thesequences of these antibodies were aligned, and a consensus sequence wasdefined by the consensus-sdAb framework sequence of the clone D10 seeSEQ ID NO:9). In addition to this llama sdAb (IsdAb) we altered thesequence so that it was more similar to human VH, an evolved consensuswas thus defined as a synthetic sdAb (hereafter referred as “hs2dAb”,see SEQ ID NO:10). We kept the specific hallmarks of VHH at four FR2positions (37, 44, 45, 47) that are conserved in conventional VH to formthe hydrophobic interface with VL and that appeared crucial forintrinsic solubility properties of sdAb (Kastelic D, et al. 2009 JImmunol Methods. October 31; 350(1-2):54-62) (FIG. 1a ). We thenconfirmed that the scaffold was robust and behaving as expected bygrafting the CDRs of a known antibody into the IsdAb and s2dAbframeworks. These experiments showed that both IsdAb and hs2dAb allowedefficient display, efficient production in bacteria and in CHO cells,and that it allowed to keep proper reactivity of the grafted CDRs bothwhen expressed in oxidative and in reducing condition. So we decided toselect the hs2dAb as a framework to construct our library.

We then introduced a synthetic diversity in the three CDRs withoutaffecting the functionality of the clones. Based on alignment ofhundreds of llama sdAb sequences we rationally designed for eachposition of the CDR1 and CDR2 a set of amino acids that still mimicnatural diversity. We voluntarily avoided cysteine residues becausethiol groups could later interfere with proper intracellular expressionand functionality. We reasoned that lowering the frequency ofhydrophobic residues or arginin would avoid aggregation (De Marco A.2011, Microb Cell Fact. June 9; 10:44 Review) and that lowering as wellproline frequency would keep most flexibility in the loops. As serine,threonine and tyrosine are the most frequent residues in CDR loopsinvolved in bonds with the epitope, aspartate and glutamate have beenproposed to increase solubility (Lodish H. et al. Molecular CellBiology. 4th edition. New York: W. H. Freeman, 2000). We voluntarilyenriched these five residues at some positions. In contrast we fullyrandomized some positions of CDR2 as well as each position of CDR3 byintroducing all amino acids except cysteine. Nevertheless a diversity inlength was also introduced in the CDR3 sequences to enrich bindingpotential to different epitope shape since nanobodies have been shown tobind both flat surface or cavity (De Genst, E., et al. Proc Natl AcadSci, (2006) March 21; 103(12):4586-91), or even haptens (Harmsen M M, etal 2007, Appl Microbiol Biotechnol., November; 77(1): 13-22. Review). Inorder to respect these statistics, to lower the occurrence of toohydrophobic residues or amino-acid promoting aggregation, and incontrast to enrich in polar aminoacid, and to further avoid theoccurrence of in frame stop or cysteins and reduce frameshift, thesynthesis of the library diversity was achieved by a unique genesynthesis technology that use double strand DNA triplet blockscorresponding to each codon (Van den Brulle J, et al. 2008.Biotechniques. September; 45(3):340-3). All codons were optimized formammalian cell expression, a Snabl restriction site was added in FR3 forfurther CDR3 loop grafting or engineering whereas any other undesiredrestriction site were avoided in the scaffold.

To generate a large complexity and to reduce the number of emptyplasmids, we constructed a novel cloning vector bearing a suicide gene(ccdB) between non compatible cloning sites. Only plasmids were thetoxic gene was lost allowed bacteria growth, hence only plasmid bearinga SDAB insert were obtained. CcdB gene is used as a positive selectionmarker. Most of the test done to detect binding activities, are doneusing a monovalent selected antibody, means of detection are then basedon anti-tag immunostaining. But monovalent tags on monovalent antibodiesdo not allow strong detection, so we decided to add a triple tag to thenovel phagemid that we constructed to increase the power of detection.

To ensure the full diversity of cloned hs2dAbs, the number oftheoretical diversity according to the rational design was far above theone of synthesized molecules which was again 4 log above the number oftransformed colonies. The most crucial was the very high moleculardiversity of hs2dAb synthesized that reached more than 10¹² sequenceswith a full probability of being unique since it was still abovetheoretical diversity imposed by the design. The fully synthetichs2dAb-L1 library was cloned in the pHEN2-3myc vector and up to 3·10⁹colonies were transformed.

The quality and functionality of the hs2dAb-L1 library has been assessedfirst by sequencing 10⁵ random clones. We then performed screeningdirected to various kind of Ag with several selection procedures. Forthe different target tested, we obtained binders with good affinitiesfor EGFP, β-Tubulin, actin, Rho conformational, p53 and Her2.Characterisation of the specificity, affinity and productivity ofselected hs2dAb binders is described.

Materials and Methods

Plasmids and Cloning

A synthetic gene (Mister Gene) composed of a 6His-Tag and a triple c-mycTag was inserted into the pHEN2 phagemid vector (Griffin I. library)between NotI and BamHI sites. The ccdB gene from pENTR™4 vector(Invitrogen) was inserted into the pHEN2 vector between NcoI and NotIsites. For mammalian expression vectors, VHH or hs2dAbs were digested byNcoI and NotI and ligated into the pAOINT or the pmCherry vectors(Clontech).

Cat Assay Filter

The pAO-CAT is a cytoplasmic expression vector that enables to fuse acarboxy-terminal HA-tagged chloramphenicol acetyl transferase (CAT) tothe VHH sequences. It has been constructed by cloning a VHH-CAT sequenceinto the pAOD-Tub1-mGFP vector (Olichon A, et al. 2007. J Biol Chem.December 14; 282(50):36314-20) digested XbaI and KpnI to remove theDsbC-Tub1-mGFP. The VHH-CAT sequence has been obtained by a multi-stepPCR strategy. The VHH was amplified using 5′CCTTGATTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGCTGATGTCCAGCTGCAGGCGT3 (Fw, SEQ ID NO:13) and 5′CCACCGCTACCGCCGCTGCGGCCGCGTGAGGAGACGGTGACCTOG 03′ (Rev, SEQ ID NO: 14). Two sequences of CATwere amplified independently using the pRill plasmid as a template toremove an internal NcoI site. For the N-term, the following primers wereused: 5′ GCGGCCGCAGCGGCGGTAGCGGTGGCGAGAAAAAAATCACTGGATATACC 3′ (Fw, SEQID NO:15) and 5′ GCCCATCGTGAAAACGGGGGCG 3′ (Rev SEQ ID NO:16).

The C-term was amplified using: 5′ CGCCCCCGTTTTCACGATGGGC 3′ (Fw, SEQ IDNO:17) and 5′AGAATAGGTACCAGCGTAATCTGGGACATCATAAGGGTAGCCACCCGCCCCGCCCTGCACTCATCG 3′ (Rev SEQ ID NO:18). The three sequences were assembledwith a final PCR and the product was digested XbaI and KpnI before beingligated into the vector. Previously selected VHHs from naïve librarywere subcloned into pAOCAT using the NcoI and Nod restriction sites.Chloramphenicol resistance assay was performed using BL21(DE3) cellstransformed with the pAOCAT-VHH fusion constructs. Bacteria were usedfor inoculating 500 μL of LB containing kanamycin (35 μg/mL) and glucose(0.2%), and were grown at 37° C. until OD₆₀₀ was 0.8. The cytoplasmicexpression of the VHH-CAT-fusion proteins was induced for 2 hours by theaddition of 0.2 mM IPTG. At the end of the induction period, bacteriaaliquots of 4 μL were plated on LB-agar plates containing IPTG (0.1 mM)and increasing chloramphenicol concentrations ranging from 0 to 500μg/ml. Bacteria were incubated at 30° C. for 20 hours beforequantification of the colony formation. The resistance level wasevaluated according to the colony growth rate at the differentchloramphenicol concentrations. Several VHH that were giving colonies upto 500 μg/ml were compared to previously characterized intrabodiesraised against GFP (nb GFP4) or Lamin (Lam) as well as to a thermostableVHH Re3 and a non intrabody C8. Liquid culture induced as above during 2hours were diluted by serial dilution and to 10 μl were spotted on agarplates containing 250 μg/ml chloramphenicol (Cam) and incubated at 30°C. for 20 hours. Colony were quantified for each dilution and normalizedto the higher amount always found with the D10 clone.

The D10 clone was further subcloned into the pHEN6 expression vector,leading to a periplasmic expression higher than 5 mg/L of culture in Ecoli X11blue strain.

It was subcloned into the pAOint-mGFP and transfected in MRC5, HEK293 orHeLa S3 cells. Transient expression of D10-GFP under the control of aCMV promoter leads to high GFP fluorescence and no aggregationdetectable compared to Lam1-GFP or Re3-GFP at 24 h and 48 h.

Library Construction

Gene collections corresponding to the FR and CDR design have beensynthesized in vitro (Sloning, GeneArt). 1 μL (10 ng) of the diversesynthesis (corresponding to 1·10¹⁰ molecules, hence 10 times the targetlibrary diversity) were amplified by PCR in a total volume of 50 μLusing 1 μL of Phusion DNA polymerase (New England Biolabs) with anequimolar mixture of the following primers:

(SEQ ID NO: 19) 5′-AACATGCCATCACTCAGATTCTCG-3′ (SEQ ID NO: 20)5′-GTTAGTCCATATTCAGTATTATCG-3′

PCR protocol consisted of an initial denaturation step at 98° C. for 45sec followed by 20 cycles of 98° C. for 10 sec, 55° C. for 30 sec and72° C. for 30 sec, and a final step extension at 72° C. for 10 min.7×150 μL of PCR were purified on 7 columns of a PCR clean-up kit(Macherey-Nagel). 55 μg of the resulting purified fragment of PCR and 80μg of the pHEN2-ccdB-3myc phagemid were digested for 2H at 37° C. withNcoI and NotI (NEB) in a total volume of 500 μL. A dephosphorylationstep was added for the phagemid with a Calf intestinal alkalinephosphatase (Sigma) 30 min at 37° C. Digestions were purified on gelwith respectively 4 and 6 columns of a gel extraction kit(Macherey-Nagel) in a final volume of 80 and 120 μL. Then, purified PCRfragment was ligated into pHEN2-ccdB-3myc, between the PelB leadersignal and the pill gene. 50 μg of phagemid and 19.2 μg of insert wereligated overnight at 16° C. with 10 μL of high concentration T4 DNAligase (NEB) in a total volume of 400 μL. Ligation was purified on 6columns (Macherey-Nagel) with a total volume of 150 μL. The ligated DNAmaterial was used to transform electrocompetent E. coli TG1 cells(Lucigen). 20 electroporations with 1 μl of ligation were performedaccording to the manufacturer's instructions (1800V; 10 μF; 600Ω). Eachelectroporation was resuspended with 1 mL of warm 2×YT, 1% glucosemedium and incubated with a shaking agitation for 1H at 37° C. 380 mL of2×YT. % glucose was added to the suspension and plated on 4302×YT-ampicillin agar dishes (140 mm) overnight at 37° C. Library sizewas calculated by plating serial dilution aliquots. The colonies werescraped from the plates with liquid 2×TY and library was stored in thepresence of 30% of glycerol at −80° C. with 1 mL aliquots at OD=38.4.3·10⁹ individual recombinant clones were obtained.

Library Sequencing:

The heterogeneity of the individual clones from the libraries waschecked by sequencing 6·10⁵ inserts on ion Torrent chips (Invitrogen).

IonTorrent sequencing library was prepared with the Ion Plus FragmentLibrary kit for AB Library Builder System (Lite Technologies) followingmanufacturer's instructions and was controlled on the Agilent 2100Bioanalyzer (Agilent Technologies) with the High Sensitivity DNA Kit(Agilent Technologies). Sequencing template was prepared by emulsion PCRwith the Ion OneTouch 2 system and the Ion PGM Template OT2 400 Kit(Life Technologies). Sequencing was performed on a IonTorrent PersonalGenome Machine using the Ion PGM Sequencing 400 Kit and a 314v2 Ion chip(Life Technologies).

Antigens

Human βActin was purchased from Sigma. RhoA GTPase fused to an aminoterminal Chitin Binding Domain or a streptactin binding peptide wereproduced in HEK293 cells. GFP in fusion with a streptavidine bindingpeptide (SBP) was produced through in vitro translation system (Roche)and used directly for screening without the need for purification(Moutel S, et al. 2009. Biotechnol J. January; 4(1):38-43). BiotinylatedTubulin was purchased from Cytoskeleton. For p53, the 72 first aminoacids of the NP_000537.3 isoform were produced in bacteria with a SNAPTag and biotinylated in vitro. For Her2, the natural receptor was usedas membrane protein target on SKBR3 cells.

Phage Display Selections

Screening for βactin, H1 histone, or FITC were performed by panning inimmunotubes as described in Marks J D et al, 1991 J Mol Biol. December5; 222(3):581-97. Screening for GFP, Tubulin and p53 were performed innative condition as described in Nizak et al. 2003 Science. May 9;300(5621):984-7. Screening on Rho was performed in native condition on atag constitutively active mutant of RhoA expressed in HEK293 cells.Screening for Her2 was performed on surface cells as described inEven-Desrumeaux K, Chames P. 2012 Methods Mol Biol.; 907:225-35.

Enzyme-Linked Immunosorbant Assay (ELISA)

Individual clones were screened as described else-where by monoclonalphage ELISA (Lee, et al; 2007, Nat Protoc. 2(11): 3001-8.

Western-Blot

After boiling in SDS-PAGE loading buffer, the samples were separated ona 12% SDS-PAGE and transferred to nitrocellulose membranes (WhatmanGmbH). Membranes were blocked in 3% non-fat milk-PBS with 0.2% Tween 20for 1 h at room temperature or overnight at 4° C. SDAB were used at1/100 and added to the membranes with an anti-hisTag antibody at 1/3000(Sigma) for 90 min. Blots were then washed and incubated 1 h withsecondary anti-Mouse HRP labeled antibodies (diluted at 1/10000 in PBS0.1% Tween 20) (Jakson ImmunoResearch Laboratories). After 5 washes withPBS 0.1% Tween 20, secondary antibodies were then revealed using theSuperSignal chemoluminescent reagent (Pierce) and Hyperfilm ECL (GEHealthCare).

Immunofluorescence

Immunofluorescence screenings were performed on HeLa cells as describedbefore (Nizak et al. 2003. Science. May 9; 300(5621):984-7).

Transient Transfection

Hela Cells cultured on coverslips were transfected according to theCaPO4 procedure with 1 μg DNA per well (24 wells plate) or 10 μg DNA (10cm2 diameter dish). Cells can be observed from 12 h posttransfection on.

Flow Cytometry

Cell surface staining were performed in phosphate-buffered saline (PBS)supplemented with 1% SFV. 100 μl of supernatant (80 μl phages+20 μlPBS/milk 1%) were incubated on 1.10⁵ cells for 1 h on ice. Phage bindingwas detected by a 1:300 dilution of anti-M13 Ab (GE healthcare) for 1 hon ice followed by a 1:1000 dilution of PE-conjugated anti-Mouse Ab (BDPharmingen.) for 45 min. Samples were analyzed by flow cytometry on aFACSCalibur using CellQuest Pro software (BD Biosciences, San Jose,Calif.).

Affinity Measurement

The binding affinity of the hs2dAb antibodies selected from the libraryand specific for GFP and ErBB2 were performed at 25° C. using a ProteOnXPR36 (BioRad) and a Biacore T200 (GE Healthcare), respectively, andfitted with a 1:1 Langmuir interaction model. The ligand GFP (24 kDa)was diluted to 1.6 μM in sodium acetate buffer (pH 5.0) and immobilizedby amine-coupling on a GLC chip (BioRad) at 730 RU. 100 μL of monovalentsingle-domain antibodies (14 kDa) were used as an analyte and injectedat 100 μL/min at concentrations between 1000 and 3 μM (60 secondinjection, 600 second dissociation). The complete kinetic set wascollected in a single run (one-shot) and, therefore, there was no needfor surface regeneration. ErbB2 ectodomain-Fc (96 kDa) was diluted to400 μg/mL in sodium acetate buffer (pH 5.0) and immobilized byamine-coupling on a CM5 chip (GE Healthcare) at 991 RU. Monovalentsingle-domain antibodies (14 kDa) were diluted in HBS-EP+ buffer andinjected as analytes at 30 μL/min at concentrations between 300 and 3 μMusing the single-cycle modality (120 second injection, 120 secondintermediate dissociation, 600 second final dissociation). The kineticswere collected in a unique sequence of injections and surfaceregeneration (10 mM glycine HCl, pH 2.5, for 30 s at 30 μL/min) tookplace only between two successive series.

Results

Library Design

In the view of making a large single domain antibody library enriched inhighly stable and functional antibody fragments, we aimed at identifyinga single VHH scaffold. We previously selected several hundreds of clonesfrom immune or naïve llama VHH libraries (Monegal A, et al. 2012 DevComp Immunol. January; 36(1):150-6). We screened a set of highlyexpressed clones using a chloramphenicol filter assay that discriminatehighly stable clone from the one prone to aggregation or unfolding inbacteria cytoplasm (Olichon). We used the pAO-CAT cytoplasmic expressionvector that enables to fuse a carboxy-terminal HA-tagged chloramphenicolacetyl transferase (CAT) to the VHH sequences. By comparison withpublished thermostable VHH (Olichon A. et al BMC Biotechnol. 2007 Jan.26:7:7) or intrabodies (Rothbauer U, et al Nat Methods. 2006 November;3(11):887-9), one scaffold, clone D10, was showing higherchloramphenicol resistance (FIG. 1a ). The D10 VHH was further fittingall our criteria of solubility, thermostability and no aggregation whileexpressed as an intrabody in mammalian cells, in the absence of anyknown antigen recognition. We then assessed if partial humanization(Vincke C, et al. J Biol Chem. 2009 Jan. 30; 284(5):3273-84) of thescaffold would affect its intrinsic properties by targeting sevenresidues that are found in human VH3. The four VHH-specific amino acidshallmarks in the framework-2 region (positions 42, 49, 50, and 52) thatappeared crucial for intrinsic solubility properties (FIG. 1c ) werekept untouched.

To test whether this scaffold may be suitable for antigen binding andused as a general scaffold for library construction, we grafted loops ofthe lam1 VHH specific of laminB protein (Rothbauer U, et al. NatMethods. 2006 November; 3(11):887-9). FIG. 2a shows that both scaffolds(IsdAbB and hs2dAb) do not perturb the display of the recombinantantibody on phages, here labelled with an anti-pill antibody. The yieldof production from culture supernatants either from E. coli or frommammalian CHO cells (Moutel S, et al BMC Biotechnol. 2009 Feb. 26; 9:14.doi: 10.1186/1472-6750-9-14) were also high and comparable for bothscaffolds, (see FIG. 2b ). The grafted synthetic antibodies were thenused to stain HeLa cells by indirect immunofluorescence. Both graftedsingle domain antibodies produced the expected staining indicating thatthey efficiently stained endogenous Lamin B (FIG. 2c ). The twosynthetic antibodies were then fused to EGFP and used as intrabodies.After transient transfection of HeLa cells, both fluorescent antibodieswere soluble and labelled their intracellular target (FIG. 2d ), as wasobserved when using the original lam1 VHH antibody (Rothbauer U, et al2006). Both scaffold, Lama and Humanized D10, grafted with lam1 CDRloops thus retained the binding property of the parental VHH. <.

Altogether, these experiments indicated that the synthetic scaffold ofhumanized D10 (herein called Synthetic Single Domain Antibody or hs2dAb)is an efficient and robust framework to display CDR loops.

Library Construction

We introduced a synthetic diversity in the three CDRs by rationallydesigning a set of amino acids that still mimic natural diversity foreach position of the CDR1 and CDR2 (based on statistical analysis ofCDRs found in published VHH binders). The amino acids residues of thesynthetic CDR1 and CDR2 have been determined by the following rules:

-   at CDR1 position 1: Y, R, S, T, F, G, A, or D;-   at CDR1 position 2: Y, S, T, F, G, T, or T;-   at CDR1 position 3: Y, S, S, S F, or W;-   at CDR1 position 4: Y, R, S, T, F, G, A, W, D, E, K or N;-   at CDR1 position 5: S, T, F, G, A, W, D, E, N, I, H, R, Q, or L;-   at CDR1 position 6: S, T, Y, D, or E;-   at CDR1 position 7: S, T, G, A, D, E, N, I, or V;-   at CDR2 position 1: R, S, F, G, A, W, D, E, or Y.-   at CDR2 position 2: S, T, F, G, A, W, D, E, N, H, R, Q, L or Y;-   at CDR2 position 3: S, T, F, G, A, W, D, E, N, H, Q, P;-   at CDR2 position 4: O, S, T, N, or D;-   at CDR2 position 5: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V,    W, K or M;-   at CDR2 position 6: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V,    W, or K;-   at CDR2 position 7: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, or    V;

The CDR3 amino acid sequence comprises 9, 12, 15 or and 18 amino acidsselected among one or more of the following amino acids: S, T, F, G, A,Y, D, E, N, I, H, R, Q, L, P, V, W, K, M.

Synthetic DNA was amplified using a low number of PCR cycles to preventthe addition of PCR-based mutations. We started the construction with2·10¹¹ different molecules. We cloned the synthetic library in the pHEN2phagemide vector modified by adding 2 supplemental myc-tags with asynthetic gene (Proteogenix) between the recombinant Ab and the pIIIgene. These additional myc-tags allow better revelation of monovalent Ab(data not shown). For the cloning we added also a suicide gene (ccdB)between NcoI and NotI that allows a positive selection of clones thathave inserted the library. A large amount of phagemid and insert wereused to obtain a lot of material to electroporate E. coli TG1 cells. 20electroporations were performed to produce the hs2dAb-L1 library.Transformed bacteria were plated on 430 large agar plates (140 mm). Thelibrary size was calculated by plating serial dilution aliquots and wasestimated to be of 3·10⁹ individual clones.

Library Sequencing:

We first evaluated the functional diversity by sequencing 315 randomclones with Sanger sequencing. 9 sequences were found either with stopcodon or with the missing of one base, one sequence missed all the CDR1,one other missed CDR1, FR1 and CDR2, and two last sequences were foundempty. Thus, only a very low number (4.1%) of defective clones wereobtained. Which suggest that the vast majority of the 3 10⁹ clones willexpress a recombinant hs2dAb.

The heterogeneity of the individual clones from the libraries wasfurther checked by sequencing 5.6·10⁵ inserts on ion Torrent chips (LifeTechnologies). The distribution of length sequences for the 4 sizes ofCDR3 was homogenous. The diversity and positional amino acids frequencywere in agreement with our design for all CDRs (data not shown). 3redundant clones were found but this observation may be linked to thePCR amplification done during the Next-Generation Sequencing procedure.

Library Screening

The hs2dAb-L1 library was screened by phage display using standardmethods (Hoogenboom H R. et al. 1998 June; 4(1):1-20) against a set ofdifferent antigens reported in Table 1. To validate the use of hs2dAb-L1in various screening approaches, we either carried out selection onbeads (which offers a large capacity of antigen presentation and keepthe antigen conformation closer to native protein), either panning onimmunotubes (referenced as standard method) or on natural antigenpresented naturally on the surface of mammalian cells (as often donewhen applying in vitro selection to therapeutic questions).

A first screening was performed in native condition (Nizak, 2005, seesupra) using biotinylated tubulin (Cytoskeleton) as a target. After tworounds of selection, 40 clones were screened at random byimmunofluorescence on HeLa cells fixed with methanol. 3 recombinant Abstained the endogenous tubulin (FIG. 3A). These 3 clones were alsousable in Western-Blotting of human protein samples (FIG. 3A). A secondscreening was performed against endogenous Her2 receptor expressed atthe surface of SKBR3 cells. A pre-adsorption of the phages was done ateach of the 3 rounds on Her2 negative MC10A cells to counter selectnon-SKBR3 specific antibodies. Using a FACS assay, 17 specificanti-SKBR3 binders were found out of 84 analysed that could be groupedin 17 non-redundant sequences. Analysis by ELISA, using Her2ectodomain-Fc (96 kDa) as a target, showed that 12 of them were directedagainst HER2 (FIG. 3B). Immunofluorescence confirmed that the antibodiesdecorated the SKBR3 plasma membrane (data not shown). A third screeningwas performed directed against the tumor suppressor p53 protein. The 72first amino acids of the NP_000537.3 isoform were produced in bacteriafused to a SNAP Tag, biotinylated in vitro and used as a target onbeads. After 3 rounds of selection, 12 clones out of 80 were foundpositive in phage ELISA (data not shown), 6 clearly labelled endogenousp53 in immunofluorence on A431 cells (data not shown) while 2 wereusable as intrabodies on HeLa cells (see FIG. 4). As expected, nostaining was observed on U2OS cells which are p53−/−. A fourth screeningwas performed to obtain conformational binder of Rho-GTP protein. Afterfour rounds of competitive selection (see materials and methods), 80unique clones were tested in ELISA on using recombinant GST-RhoAproteins loaded with GDP of the non-hydrolysable analogue GTPγS. 24antibodies gave a stronger signal against GTPγS-RhoA than againstGDP-Rho, indicating conformational binding. One of the antibodies, theH12 clone, dominated the screen and was the only clone obtained upon anadditional 5th round of selection. We tested these conformationalbinders in immunofluorescence on HeLa cells expressing SF-GFP(superfolder GFP) fusion of RhoA negative mutant N19 (GDP-bound) or theconstitutively active mutant L63 (GTP-bound). None of the clones stainedcells expressing the N19 negative mutant while 8 of them efficientlystained cells overexpressing the active form of RhoA. We furthercharacterized the clone H12 and tested its ability to pull downendogenous RhoA from HeLa cell extracts incubated with GTPγS or GDP. Weshowed that purified H12 antibody was an effective conformationalbiosensor of Rho activity usable in ELISA, immunofluorescence and invitro pull down experiments. We further observed that it was moreeffective than the conventional RBD activity assay were the Rho BindingDomain of an effector protein is used as a biosensor. (FIG. 3C, IF andELISA Data not shown). A fifth screening in native conditions wasperformed using the GFP protein as a target. Thirty seven non redundantclones out of 80 analysed were positive in phage ELISA (data not shown).Ten of them detected GFP-myr-palm, by immunofluorescence (data notshown). Importantly, we observed that 4 of these antibodies were usableas intrabodies against recombinant GFP expressed in Hela cells (FIG. 4).A last screen was performed using to immunotubes coated with nativecommercial bActin (Sigma). This last screening allowed the selection of16/S0 unique binders as observed by phage ELISA (data not shown), Sevenof them clearly decorated endogenous actin stress fibers in HeLa cells(data not shown) and 4 were usable for Western-Blotting experiments(data not shown).

TABLE 1 Summary of screening Positive clones Phage Rounds of AntigenELISA IF/FACS Intrabody punning GFP 37 10/   4/10 2 mCherry ND 6/ 2/6 3Tubulin ND 3/ 0/3 2 Actin 11 9/ 1/7 3 p53 12 6/ 2/6 2 RhoA-GTP 24 8/ 3/84 Her2 15  5/10 ND 3

Affinity Measurement

Affinity of single domain antibodies against GFP, Her2 and p53 weremeasured using a ProteOn XPR36 (BioRad) or a Biacore T200 (GEHealthcare). Affinities were estimated to be in the nanomolar range:3.06 10⁻⁸ M for anti-GFP, 1.94 10⁻⁸ M for anti-Her2 and 3.25 10⁻⁸ M foranti-p53, demonstrating that high affinity binders could be obtainedfrom our hs2dAb-L1 library of synthetic, non immune, single domain Ab(FIG. 5).

Inhibitory Intrabodies

Identification of blocking antibodies is a challenging task. However, itis possible to functionalize non-blocking intrabodies to inhibit theirtarget function. One approach relies on the ubiquitinylation anddegradation of the recognized target as described by Caussinus et al.2011 (Caussinus et al. 2011, Nat. Struct. Mol. Biol. 19, 117-121). Thisapproach is based on the fusion of intrabodies to an F-box domain whichallows interaction with Skip1, a member of the SCF complex, an E3ubiquitin ligase of the complex E1/E2/E3 ubiquitinylation machinery,that target proteins to proteasome-dependent cellular degradation. Thisapproach was initially developed to target several GFP fusion proteinsin Drosophila using a single anti GFP intrabody, named GFP4, which is arobust high affinity GFP llama intrabody originally isolated from animmune library (Rothbauer, U. et al. Nat. Methods 3, 887-889). To getinsight into the relative functionality of hs2dAb for such a proteinknockdown approach, we fused several of our anti GFP hs2dAb at theiramino terminus to the Fbox domain and compared their efficacy with theefficacy of the Fbox-GFP4 antibody. To detect cells expressingFbox-intrabody fusion proteins (F-Ib), we constructed a bicistronicvector driving the co-expression of F-Ib together with amitochondria-targeted mCherry (Mito-mCherry). We expressed the F-Ibantibodies in a HeLa clone stably expressing GFP fused to histone H2B(Silljé, H. H. W., Nagel. S., Kömer, R. & Nigg, E. A., 2006, Curr. Biol.CB 16, 731-742) and looked for GFP-H2B depletion. As expected, F-GFP4,also known as degradFP, induced a strong reduction of H2B-GFP expressionas analyzed by western blot (data not shown). Accordingly, a strongreduction in nuclear fluorescence intensity was observed in cellsexpressing F-GFP4. No effect was observed % when expressing either GFP4alone or a GFP4 fused to a truncated, nonfunctional, Fbox domain. Whenwe tested anti-GFP clones selected from our novel library, we observedthat several hs2dAb that were found to be efficient when used asfluorescent intrabodies failed to degrade H2B-GFP when expressed asF-Ib. This highlights the fact that not all intrabodies can efficientlybe functionalized with the F-box. However, one hs2dAb anti-GFP induced acomplete disappearance of nuclear H2B-GFP signal when expressed as F-Ib.FACS analysis showed a fluorescence intensity decreased as much as 70%.As expected, this effect was reversed in the presence of proteasomeinhibitor treatment.

Altogether, these experiments show that the hs2dAb scaffold enables thefrequent selection of antibodies that can be expressed in the mammaliancell cytoplasm to be used as fluorescent intrabodies or inhibitoryintrabodies.

Useful Sequences for practicing the invention SEQ ID NO: DescriptionSequence 1 single domain VQLQASGGGFVQPGGSLALSCAASG antibody FR1 2single domain MGWFRQAPGKEREFVSAIS antibody FR2 3 single domainYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTATY antibody FR3 YCA 4 single domainYWGQGTQVTVSS antibody FR4 5 coding nucleic acidATGGCGGAAGTGCAGCTGCAGGCGAGCGGCGGCG sequence of SEQ ID GCTTTGTGC NO: 1AGCCGGGCGGCAGCCTGCGTCTGAGCTGCG CGGCGAGCGGC 6 coding nucleic acidATGGGCTGGTTTCGTCAGGCGCCGGGCAAAGAACG sequence of SEQ ID TGAATTTG NO: 2TGAGCGCGATTAGC 7 coding nucleic acidTATTATGCGGATAGCGTGAAAGGCCGTTTTACCATT sequence of SEQ IDAGCCGTGATAACAGCAAAAA NO: 3 CACCGTGTATCTGCAGATGAACAGCCTGCGTGCGGAAGATACCGCTACGTATTATTGCGCG 8 coding nucleic acidTATTGGGGCCAGGGCACCCAGGTGACCGTGAGCAG sequence of SEQ ID CGCGGCCGCA NO: 49 Non humanized VQLQESGG GFVQAGGSLR LSCAASGFTF sequence of cloneSSYAMGWFRQ APGKEREFVA D10 AISDSSGNHAYVADSVKG RFTISRDNAKNTVYLQMNSL KPEDTATYYC ARSDAAGNPS GYWGQGTQVTVSS 10 Humanized sequenceVQLQASGG GFVQPGGSLR LSCAASGFTF of clone D10 SSYAMGWFRQ APGKEREFVSAISDSSGNHAYVADSVKG RYTISRDNSK NTVYLQMNSL RAEDTATYYC ARSDAAGNPSGYWGQGTQVTVSS 11 oligonucleotide 5p CCCACCAAACCCAAAAAAAGAGATCCTAGAACTA8328 GCTATGGCCGGACGGGCCATGGCGGAAGTGCAGCT GCAGGCTTC 12 oligonucleotide 3pACCGGGCCTCTAGACACTAGCTACTCGAGGGGCCC 8329CAGTGGCCCTATCTATGCGGCCGCGCTACTCACAG TTAC 13 VHH Fw primerCCTTGATTCTAGAAATAATTTTGTTTAACTTTAAGA AGGAGATATACCATGCTGATGTCCAGCTGCAGGCGT 14 VHH Rev primerCCACCGCTACCGCCGCTGCGG CCGCGTGAGGAGAC GGTGACCTGG G 15 Fw N-termGCGGCCGCAGCGGCGGTAGCGGTGGCGAGAAAAA AATCACTGGATATACC 16 Rev N-termGCCCATCGTGAAAACGGGGGCG 17 Fw C-term CGCCCCCGTTTTCACGATGGGC 18 Rev C-termAGAATAGGTACCAGCGTAATCTGGGACATC ATAAGGGIAGCCACCCGCCCCGCCCTGCACTCATC G 19Primer library AACATGCCATCACTCAGATTCTCG 20 Primer libraryGTTAGTCCATATTCAGTATTATCG

1. A method of making a synthetic single domain antibody library, saidmethod comprising i. introducing a diversity of nucleic acids encodingCDR1, CDR2, and CDR3, between the respective framework coding regions ofa synthetic single domain antibody to generate nucleic acids encoding adiversity of synthetic single domain antibodies with the same syntheticsingle domain antibody scaffold amino acid sequence, wherein saidsynthetic single domain antibody scaffold comprises the followingframework regions consisting of FR1 of SEQ ID NO: 1, FR2 of SEQ ID NO:2,FR3 of SEQ ID NO: 3 and FR4 of SEQ ID NO:4, or functional variantframework regions with no more than 1, 2 or 3 conservative amino acidsubstitutions within each framework region. 2-4. (canceled)
 5. Themethod according to claim 1, wherein the amino acid residues of thesynthetic CDR1 and CDR2 of at least 70%, 80% or at least 90% of theclones of the library, are determined by the following rules: at CDR1position 1: Y, R, S, T, F, G, A, or D; at CDR1 position 2: Y, S, T, F,G, T, or T; at CDR1 position 3: Y, S, F, or W; at CDR1 position 4: Y, R,S, T, F, G, A, W, D, E, K or N; at CDR1 position 5: S, T, F, G, A, W, D,E, N, I, H, R, Q, or L; at CDR1 position 6: S, T, Y, D, or E; at CDR1position 7: S, T, G, A, D, E, N, I, or V; at CDR2 position 1: R, S, F,G, A, W, D, E, or Y; at CDR2 position 2: S, T, F, G, A, W, D, E, N, H,R, Q, L or Y; at CDR2 position 3: S, T, F, G, A, W, D, E, N, H, Q, P; atCDR2 position 4: G, S, T, N, or D; at CDR2 position 5: S, T, F, G, A, Y,D, E, N, I, H, R, Q, L, P, V, W, K or M; at CDR2 position 6: S, T, F, G,A, Y, D, E, N, I, H, R, Q, L, P, V, W, or K; at CDR2 position 7: S, T,F, G, A, Y, D, E, N, I, H, R, Q, L, P, or V; and wherein CDR3 amino acidsequence comprises between 9 and 18 amino acids randomly selected amongone or more of the following amino acids: S, T, F, G, A, Y, D, E, N, I,H, R, Q, L, P, V, W, K, M.
 6. A synthetic single domain antibody libraryobtainable by the method of claim
 1. 7. The synthetic single domainantibody library of claim 6, comprising at least 3·10⁹ distinct antibodycoding sequences. 8-9. (canceled)
 10. An antigen-binding protein,comprising a synthetic single domain antibody of the following formula:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein said framework regions consistof FR1 of SEQ ID NO:1, FR2 of SEQ ID NO:2, FR3 of SEQ ID NO: 3 and FR4of SEQ ID NO:4, or, their functional variants with no more than 0, 1, 2or 3 conservative amino acid substitutions in each of FR1, FR2, FR3 andFR4.
 11. (canceled)
 12. The antigen-binding protein of claim 10, whereinsaid synthetic single domain antibody has one or more of the followingfunctional properties: i. it can be expressed as soluble single domainantibody in E. coli periplasm, ii. it can be expressed as solubleintrabodies in E. coli cytosol, iii. it is stable in reducingenvironment in chloramphenicol acetyl transferase fusion assay, iv. itdoes not aggregate when expressed in mammalian cell lines as fluorescentprotein fusions. 13-14. (canceled)
 15. The antigen-binding protein ofclaim 10, wherein the amino acid residues of the synthetic CDR1 and CDR2are: at CDR1 position 1: Y, R, S, T, F, G, A, or D; at CDR1 position 2:Y, S, T, F, G, T, or T; at CDR1 position 3: Y, S, F, or W; at CDR1position 4: Y, R, S, T, F, G, A, W, D, E, K or N; at CDR1 position 5: S,T, F, G, A, W, D, E, N, I, H, R, Q, or L; at CDR1 position 6: S, T, Y,D, or E; at CDR1 position 7: S, T, G, A, D, E, N, I, or V; at CDR2position 1: R, S, F, G, A, W, D, E, or Y; at CDR2 position 2: S, T, F,G, A, W, D, E, N, H, R, Q, L or Y; at CDR2 position 3: S, T, F, G, A, W,D, E, N, H, Q, P; at CDR2 position 4: G, S, T, N, or D; at CDR2 position5: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, K or M; at CDR2position 6: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, V, W, or K; atCDR2 position 7: S, T, F, G, A, Y, D, E, N, I, H, R, Q, L, P, or V; andwherein CDR3 amino acid sequence comprises between 9 and 18 amino acidsselected among one or more of the following amino acids: S, T, F, G, A,Y, D, E, N, I, H, R, Q, L, P, V, W, K, M.
 16. The antigen-bindingprotein of claim 10, further comprising a F-box domain for targeting aprotein to the proteasome.
 17. An isolated nucleic acid that encodes anantigen-binding protein comprising a synthetic single domain antibody ofthe following formula: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein saidframework regions consist of FR1 of SEQ ID NO: 1, FR2 of SEQ ID NO:2,FR3 of SEQ ID NO: 3 and FR4 of SEQ ID NO:4, or, their functionalvariants with no more than 0, 1, 2 or 3 conservative amino acidsubstitutions in each of FR1, FR2, FR3 and FR4.
 18. The isolated nucleicacid of claim 17, comprising the following nucleic acid sequences: SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, encoding respectivelyframework regions FR1, FR2, FR3 and FR4 of SEQ ID NOs 1-4, or variantcorresponding sequences with at least 90% identity to said SEQ ID NOs5-8, encoding functional variants of FR1, FR2, FR3, and FR4 of SEQ IDNOs 1-4.